The principal aim of this research is to study the nucleolytic processing of mitochondrial DNA of mammalian cells. The potent endonuclease of bovine mitochondria which likely plays a major role in degradative processing of DNA in vivo will be further characterized and purified. Recently, the endonuclease has been shown to degrade DNA in a nonrandom fashion, preferring attack in tracts of guanine residues. Strong cleavage sites in the D-loop region of bovine mt DNA will be further mapped and factors (pH, salt, divalent cations, temperature, etc.) will be surveyed to determine if cleavage can be restricted one specific site. Cleavage specificity with ss DNA and mRNA templates will also be studied. A mt DNA binding protein has recently been identified which protects ss DNA from degradation in vitro. This protein will be purified extensively and characterized, and its ability to protect duplex DNA and mRNA from degradation will also be examined. In addition, processivity of the endonuclease, capacity to selectively process D-loop DNA and potential to recognize apurinic sites in DNA will be studied. Guanine nucleotides and oligonucleotides will be checked for a specific inhibitory effect on enzyme activity. Murine monoclonal antibodies will be prepared and an immunoabsorptive method will be developed to remove the nuclease from mt lysates. The intracellular and tissue distribution of the endonuclease will be quantitated and characterized. Efforts to reconstitute an in vitro DNA replication assay will be started. Initially, this work will focus on enzymes which recognize the H-strand origin (OH) to initiate and synthesize the nascent H-strand of the D-loop. The mt RNA polymerase and DNA polymerase-Gamma will be further purified and characterized. The presence of this nuclease in soluble extracts of mitochondria has greatly impeded past efforts to reconstitute a variety of mt DNA replicative reactions in vitro. This nuclease problem is unfortunate because mt DNA would otherwise appear to be an attractive well-defined template probe to study the enzymology and mechanism of DNA replication in higher cells. This study will try to develop an effective method to specifically remove or inhibit the nucleolytic activity.